Retroviruses are RNA viruses which cause a large variety of different tumors in many species of vertebrates. The long-term goal of this proposal is to understand in molecular detail each step in the replication of the Moloney murine leukemia virus, one of the most intensively studied of the retroviruses. Specifically, we will determine the function of many of the viral gene products, the time in the life cycle when they are needed, and the sites on the viral genome with which they interact. A major effort will be mounted to determine the nature of the gene products involved in the integration of the viral DNA into the host chromosome. A combination of genetic and biochemical approaches will be used to achieve these goals. Site-directed mutagenesis of cloned viral DNAs will be used to create alterations at specific positions in the genome; mutant viruses can then be isolated by cotransformation of murine cells in culture with the altered DNAs. These viruses will be analyzed to determine the step blocked by the mutation, allowing functions to be assigned to each viral gene. The protein products of the viral pol gene will be directly analyzed for DNA binding and endonuclease activities; these proteins will be prepared in large quantity by forcing the expression of portions of the pol gene in E. coli. The sites at which these proteins act will be probed by mutagenesis of the tips of the viral DNA. Finally, we will use one set of our mutants to study the mechanisms of homologous recombination used by the retroviruses to exchange genetic information and to acquire new sequences from the host. These studies will provide basic information about the replication of the retroviruses, and especially the means by which recombination with cellular DNA occurs. This information is essential to our understanding of the pathogenicity of these viruses, and is likely to give new insights into the important processes of recombination and transposition carried out by mammalian cells.